Transcript 11: GPC
ADVANCED BIO-FRIENDLY POLYMERS Gel permeation chromatography – a tool for determination of molar mass of polymers
Igor Lacík
Molecular weight: characterization techniques
M
n techniques related to colligative properties (dependence on the number of molecules) membrane osmometry (> 25 000 g/mol) vapor pressure osmometry (< 25 000 g/mol) mass spectrometry electrospray ionization mass spectrometry (ESI-MS) matrix-assisted laser desorption/ionization (MALDI) size exclusion chromatography
M
w static laser light scattering analytical ultracentrifuge size exclusion chromatography
M
z static laser light scattering analytical ultracentrifuge size exclusion chromatography
M
n
M
w
M
z
M
i
M
viscometry size exclusion chromatography
Molecular weight: characterization techniques
M
n techniques related to colligative properties (dependence on the number of molecules) membrane osmometry (> 25 000 g/mol) vapor pressure osmometry (< 25 000 g/mol) mass spectrometry electrospray ionization mass spectrometry (ESI-MS) matrix-assisted laser desorption/ionization (MALDI) size exclusion chromatography
M
w static laser light scattering analytical ultracentrifuge size exclusion chromatography
M
z static laser light scattering analytical ultracentrifuge size exclusion chromatography
M
n
M
w
M
z
M
i
M
viscometry size exclusion chromatography
Size-exclusion chromatography – principle
1. Column separation technique 2. Based on
enthalpy-free partitioning
of analyzed solutes, most often the polymer chains, of different molecular weight / length (size) between mobile and stationary phases
Separation mechanism is based on the size
• • •
Stationary phase pore size pore size distribution particle size
•
Mobile phase
flow rate in ml/min providing “elution volume, V e ” or elution time, t e ”
Separation mechanism is based on the size
Time 3 Time 2 Time 1
Time 1 < Time 2 < Time 3
V
e,1
< V
e,2
< V
e,3
Size-exclusion chromatography – principle
1. Column separation technique 2. Based on
enthalpy-free partitioning
of analyzed solutes, most often the polymer chains, of different molecular weight / length (size) between mobile and stationary phases
V e
V
0
K d
(
V t
V
0 ) K d - partition coefficient 0 K d 1 solute exluded solute permeated V e - elution volume for given size V 0 - interstitial volume (between particles of column packing) V t - total available volume of column (interstitial and pore volumes)
Size-exclusion chromatography – setup
Injection of polymer
of unknown molecular weight characteristics
Pump
eluent
Commercial columns of defined characteristics for column packing:
diameter, exclusion limit, pore-size distribution
Detector signal = f(elution volume)
calibration curve with standards:
elution volume = f(MW)
molecular weight characteristics (MWD, M w , M n ) of polymer molecular weight distribution
exclusion separation permeation log M
GPC signal
w
(log
M
) ln( 10 )
M f w
(
M
) ln( 10 )
M
2
f n
(
M
)
Size-exclusion chromatography – hardware
PUMP INJECTOR COLUMNS MALLS DEGASSER RI SOFTWARE MAREK Degasser Rheodyne inj . or Waters Autosampler Pump Waters 515 Columns MALLS SLD7000 DRI Detector Waters 2410 WinGPC®7.4 Software
Size-exclusion chromatography – calibration SEC is an indirect method
needs RELATION of elution volume (volume slice) to molecular weight = calibration • several types of calibration 1. narrow distributed standards of the same polymer
M w /
[ ] 1
M
M
n
1 = [ 1.1
] 2 M 2 ~ size of coil ~ V elution (Benoit et al, 1966) note: a) Mark-Houwink equations is related to MW range b) careful in low MW range where a ~ 0.5 J.Polym.Sci. 6, 1759 (1968) 3. absolute molecular weight detectors (LS, viscosity) 4. effective calibration (no match between standards and analyzed polymer) decrease in accuracy
Size-exclusion chromatography – hints/experience
Remember suppress interactions to avoid non-SEC phenomena
(1)
polymer - polymer (aggregation) – earlier elution, artificially increased molecular weight
(2)
polymer - column packing (adsorption) - delayed elution, artificially decreased molecular weight
(3)
eluent-column packing (repulsion) – earlier elution, artificially increased molecular weight
(4)
polymer - eluent (aggregation) - earlier elution, artificially increased molecular weight
(5)
etc.
Size-exclusion chromatography – hints/experience
Remember suppress interactions to avoid non-SEC phenomena
water-soluble polymers (polyelectrolytes)
Problem •H-bonding •hydrophobic interactions •polyelectrolyte effect •adsorption (ion inclusion) •repulsion (ion exclusion) •calibration •concentration effect •resolution (eluent viscosity) Cure Eluent composition & column selection PSS TosoH Biosep Polym. Laboratories Waters Suprema HEMA GRAM (DMAc) TSK PWXL Ultrahydrogel Direct calibration with standards of well-defined M p Optimized solute concentration Temperature of analysis
Size-exclusion chromatography – hints/experience
Remember suppress interactions to avoid non-SEC phenomena
water-soluble polymers (polyelectrolytes like poly (acrylic acid))
eluent: 0.1M LiNO columns: 3 , 0.04 M phosphate bufer, pH 8.0, 0.01M NaN Suprema 100, 1000, 3000, 10 00 data acquisition and evaluation 0 Å (8 x 300 mm, 10 3 m m) flow rate 0.6 -1.0 ml/min (ethylene glycol – internal standard) temperature 60-80ºC calibration p(NaA) standards 1 250 – 1 100 00 0 g/mol (PSS, Mainz, Germany) sample: concentration 0.5 - 2 mg/ml, injection volume 200 m L WinGPC 7 (PSS, Mainz, Germany) or 0.1M Na 2 HPO 4 (pH 9) or other compositions EG system peaks - salt excluded from PE region sample
Size-exclusion chromatography – hints/experience
Remember suppress interactions to avoid non-SEC phenomena Calibration curve (polyacrylic acid) Guide-lines: • no upward curvature at high M.w, i.e. no adsorption of PAA to column packing (solute-gel interactions) pronounced at M.w.
• variation of ionic strength - test to estimate the interactions ionic strength too low: elution volumes increase due to electrostatic interactions ionic strength too high: elution volumes increase due to hydrophobic interactions remove charged monomer by dialysis: as it adds to the ionic strength of eluent
a suitable ionic strength is in case of the lowest elution volumes
Size-exclusion chromatography – hints/experience
Remember suppress interactions to avoid non-SEC phenomena 0.4
0.3
0.2
0.1
0.0
0.8
0.7
0.6
0.5
1.1
1.0
0.9
PAA concentration in mg/ml Concentration effect (polyacrylic acid) 5.0
22.0
8E+5 6E+5 3.0
21.0
4E+5 1.0
0.7
0.3
10 3 10 4 Mol mass 10 5 10 6 g/mol 20.0
0 1 2 3 4 5 decrease in hydrodynamic volume of polymer coil at higher polymer concentration leads to the artificially lower M.w.
lowering the sample concentration until the constant molar mass is reached yet detector sensitivity is sufficient 6 2E+5
Size-exclusion chromatography – hints/experience
Accuracy of the flow rate (polyacrylic acid) ! EXTREME SENSITIVITY TO VARIATION IN THE FLOW RATE ! Elution volume of internal standard [ml] Difference [%] M p [g/mol] Difference in M p [%] sample EG
36,525
36,342 36,160 35,794 36,707 36,890 37,256
0
- 0,5 - 1,0 - 2,0 + 0,5 + 1,0 + 2,0
57 700
55 000 50 700 42 600 64 300 69 300 80 000
V adjusted sample
V analysis sample V calibratio
int .
s
tan
n dard V analysis
int .
s
tan
dard
it is essential flow rate is controlled by the flowmarker (ethylene glycol) which also controls the pore volume and any changes on columns
0
- 4,7 -12,1 -26,2 11,4 20,1 38,6
Size-exclusion chromatography – hints/experience
Effect of temperature of analysis (polyacrylic acid) 2.0
1.5
1.0
0.5
room T Water Toluene THF 0.0
0 20 40
Temperature [ C]
60 80 the partitioning of the solute between mobile and stationary phases depends on viscosity of mobile phase: improved partitioning improved resolution viscosity of aqueous mobile phase can be adjusted to that of the organic one by the temperature of analysis
Size-exclusion chromatography – hints/experience
Final hints with special emphasis to the aqueous-phase SEC • fast advancing in the last years with strong achievements (columns and new experience) • there exist more and more examples of very precise analysis with high resolution (a strong improvement in aqueous-phase SEC) • patience in thorough testing after thinking of all the possibilities which may hamper the experiment • column history may play a role (more in aqueous-phase SEC than in aqueous-phase SEC)
Size-exclusion chromatography – final slide
This is also the common SEC analysis